The epidemiological success of IAV relies on airborne transmission from person-to-person; however, the viral properties governing airborne transmission of IAV are complex. Receptor-binding specificity is an important determinant of host-range restriction and transmission of IAV. The ability of zoonotic IAV for airborne transmission increases their pandemic potential. IAV infection is mediated via binding of the viral hemagglutinin (HA) to terminally attached 2,3 or 2,6 sialic acids (SA) on cell surface glycoproteins. Human IAV preferentially bind 2,6-linked SA while avian IAV bind 2,3-linked SA on complex glycans on airway epithelial cells. Historically, IAV with preferential association with 2,3-linked SA have not transmitted efficiently by the airborne route in ferrets. We used an epidemiologically successful IAV in which we altered receptor preference from the human (2,6SA) to the avian receptor (2,3SA) and observed efficient airborne transmission. Airborne transmission was associated with rapid selection of virus with a change at a single HA site which conferred binding to long-chain 2,6SA, without loss of 2,3SA binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours post-infection and was remarkably enriched in the soft palate, where long-chain 2,6SA predominate on the nasopharyngeal surface. Importantly, presence of long-chain 2,6SA is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled, rapidly selects for transmissible IAV with human receptor (2,6SA) preference. Vaccination remains the primary strategy for the preventing severe influenza. Owing to antigenic drift in circulating viruses, a vaccine from one season may not be effective in subsequent seasons, so annual immunization is needed to maintain immunity. Each year, the strains that are to be included in the vaccine for the next influenza season are chosen by the WHO collaborating centers. With the view of improving live vaccine efficacy, reducing the time of vaccine production, and limiting the interference of pre-existing immunity to the vaccine backbone, novel approaches to vaccine development are being investigated. Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA+NA)Min ), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA+NA)Min virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (106 TCID50 /ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA+NA)Min virus at doses ranging from 103 to 105 TCID50 led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA+NA)Min virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.